Mutation discovery is one of the most important issues for
the gene-driven ENU mutagenesis. For the high-throughput search
of point mutations, we have adopted the Temperature Gradient
Capillary Electrophoresis (TGCE) method. It is relatively low
cost and high-throughput for mutation discovery. By using
positive controls, we calibrated the running condition of TGCE
for several target amplicons. We have so far screened 24 genes
in 6000 G1 mice by TGCE and succeeded in finding novel 50
point-mutations in 19 genes. A few mutations were found to be
identical probably due to the clonal expansion during the
mutagenized spermatogenesis. Subtracting these duplicates, we
concluded that there are 46 independent ENU-induced mutations
by screening the total of 60 Mbp. Out of 24 genes screened, we
have not been able to detect any mutations in 5 genes and have
found only one mutation in the other 5 genes. The remaining 14
genes have been identified more than one mutation. The mutation
rate of one in 1.3 Mb by TGCE seems to be lower than that
obtained by the direct-sequencing method of one in
approximately 1 Mb. In spite of somewhat lower detectability by
using TGCE, the gene-driven approach concretely detect point
mutations in the target genes. In most of the cases, multiple
independent mutation alleles have been found so that it now
becomes feasible and practical to study the functions of the
genes from domain to domain separately. Recently we are
screening about 8-9 Mb and finding 6 mutations on average per
month.